A biomarker is a characteristic that may be measured and evaluated as an indicator of the biological state of an organism. In medicine, a biomarker may be an exogenous substance that is introduced into a patient to examine biological processes, such as organ function, and biochemical functions and pathways. A biomarker may also be a biomolecule obtained from a patient, such as a protein or a nucleic acid, that indicates a particular disease state or response to a drug therapy. Such biochemical biomarkers are also particularly useful in the discovery and development of new drugs. During early phase clinical research of such drugs, quantification of suitable biomarkers may potentially aid researchers in more rapid identification of the most promising drug candidates, thus streamlining the drug development process. Disease-related biomarkers may also be used for diagnosis or prognosis of disease, and as a measure of therapeutic efficacy. Thus, it is of great importance to identify and validate new biomarkers that may be of use in assessing patient health and/or response to therapeutic interventions, as well as provide a method for accurate and reproducible determination of known biomarkers.
Biomarker analysis is highly dependent on the integrity of reagents such as antibodies, which are themselves derived from biologic sources and thus may be subject to issues of quality control and stability. In many biomarker assay processes, non-certified standards, such as recombinant proteins and surrogate matrices have been used, in order to derive a calibration curve. Thus, parallel studies need to be performed where the response of the assay to a range of calibration standard concentrations made up in the surrogate matrices is comparable to that of a series of dilutions of patient samples. Dilution linearity can also be problematic, as antibody and ligand-binding affinities can vary significantly in different media. The goal of biomarker assay development and qualification is to develop assays for clinical benefit.
Immunoassays such as the Enzyme Linked Immunosorbent Assay (“ELISA”), based on the binding specificity of antibodies for a target antigen, are well-known in the art (see for example, Engvall et al., Immunochem. 1971, 8:871; Ljunggren et al., J. Immuno. Meth. 1987, 88:104; Kemeny et al., Immunol. Today 1986, 7:67). Immunoassays are highly useful for identifying new biochemical biomarkers such as proteins or nucleic acids, as well as quantifying known biomarkers, as antibodies can be generated against a specific biomarker.
Examples of methods of quantitative determination of biomarkers via immunoassays are known in the art. See for example, Hawkes et al., Anal. Biochem. 1982, 119:142-147, and Tobin and Gordon, J. Immunol. Methods 1984, 72:313-340, and U.S. Pat. No. 5,486,452 to Gordon et al., entitled “Device and kits for immunological analysis”.
Previously employed immunoassay methods tended to be limited as they could only detect one target analyte per test cycle, within a single reaction vessel. Attempts have been made to decrease the time for completion of each test cycle, and to increase the number of tests that are carried out per cycle, by adhering probe molecules (e.g. antibodies) to a solid substrate (e.g. a bead or a well on a plate), and then washing test samples, buffers, and reagent solutions over the solid substrate.
A microarray is a device in which a large number (e.g. hundreds to thousands) of samples of biomolecules, such as DNA and proteins, are affixed or immobilized to a suitable non-reactive substrate surface, such as plastic (e.g. polypropylene, polystyrene, cyclo-olefins), silicone, and glass. If the substrate surface is relatively flat, the biomolecules may be “printed” on the surface, whereby printing is carried out by application of a known volume of a “spotting” buffer containing a known concentration of the biomolecule. With the biomolecules fixed to a known substrate or known locations on a substrate surface, the substrate surface may then be exposed to biochemical/chemical reagents for the purposes of detection, and qualitative and quantitative analysis. For example, a microarray may be used to carry out an ELISA-type immunoassay, wherein the biomolecule affixed to the substrate is an antibody, and the substrate surface is then exposed to a test solution containing an antigen to which the antibody can bind. The substrate surface can then be washed with a buffer solution and exposed to a secondary antibody which is conjugated to either a detectable label (e.g. a radiolabel or a fluorescent dye) or an enzyme which catalyzes a reaction for which the reaction product is coloured and thus detectable. The microarray thus allows high throughput analysis of large quantities of samples. At the same time, computer software programs have been developed to analyse the large quantities of data that are generated from a microarray.
A number of examples of microarrays and methods of handling the data provided by microarrays are known in the art. See for example, U.S. Pat. No. 6,516,276 to Ghandour et al., entitled “Method and apparatus for analysis of data from biomolecular arrays”, U.S. Pat. No. 6,916,621 to by Shah, entitled “Methods for array-based comparative binding assays”, and U.S. Pat. No. 7,072,806 to Minor, entitled “Methods and systems for comparing data values across multiple platforms”.
It is typical for several antigenic substances or biomarkers to be associated with the detection and diagnosis of a biological process, including diseases. To confirm the presence of multiple biomarkers, each marker within a test sample would require a separate immunoassay to be carried out. This greatly increases the amount of time to analyse a given test sample, and gives rise to other problems, such as increased cost and increased experimental errors which increase with every assay that must be carried out. Thus, it is desirable to identify and employ methods of quantitative determination of biomarkers that allow detection and quantitative measurement of multiple antigens or biomarkers simultaneously, i.e. “multiplex” detection and determinations.
As a microarray allows simultaneous, multiple biochemical analyses, microarrays may be adapted to perform multiplex analyte detection. In order to increase the capacity of existing microarrays, “multiplex” microarrays have been developed, wherein multiple different probe biomolecules are present in the same microarray. This allows users to detect and analyse more than one target analyte in a test sample. This is particularly useful for high-throughput screening of tissue/body fluid samples for multiple biomarkers that may be used for detection and diagnosis of biological processes including pathogenic and physiological disorders.
There are a number of problems that may arise during microassays, including difficulties in obtaining accurate quantitative analyses and reproducibility. Such problems tend to be magnified in attempts to carry out multiplex detections. Cross-hybridization may occur between biomolecules that have been fixed to the surface of the microarray. In addition, not all the desired amount of biomolecule may adhere to the substrate surface during the printing process. For example, in the case of a relatively flat substrate surface, there may be an uneven printing of the amounts of biomolecule on the substrate surface, which would affect accuracy in quantitative analyses. Furthermore, during the course of an assay, unknown amounts of the biomolecules may be washed away during the application and removal of assay reagents. Thus, the actual amount of the biomolecule within a given location on a substrate surface during the course of the assay may be less than the theoretical printed amount, i.e. the amount calculated based on the known concentration of the spotting buffer and the volume of spotting buffer applied to the surface. However, prior art methods for quantifying analyte amounts in a test sample do not take into account the discrepancies between the theoretical amounts and the actual amounts of the biomolecule immobilized on the microarray.
Accordingly, there is a need to develop a method to normalize the data obtained from a microarray to minimize the experimental errors inherent to the microassay method, such as may be due to discrepancies between the theoretical concentration of a biomolecule on the microarray substrate surface, based on the concentration of the coating/spotting buffer, and the actual concentration of the biomolecule that exists on the substrate surface. In addition, as a single test cycle of a multiplex microarray may provide data from multiple assays or batches of standards, there is a need for a method to compare multiple assays or batches of standards to enable conversion values between different assays or standards and to provide a reference basis across all such assays.